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Enhancing the circulating half-life of antibody-based fusion proteins

a fusion protein and circulating half-life technology, applied in the field of fusion proteins, can solve the problems of limited use of recombinantly-produced antibody-based fusion proteins, achieve enhanced in vivo circulating half-life, decrease and increase the interaction of fc moiety

Inactive Publication Date: 2006-02-16
MERCK PATENT GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent text describes a method for improving the half-life of fusion proteins containing an immunoglobulin and a non-immunoglobulin protein. The method involves altering certain amino acids near the junction of the two proteins. This alteration can lead to a significant increase in the serum half-life of the fusion protein, even when the interaction with Fc receptors and Fc protection receptors is not the main factor determining the half-life. The altered amino acids can affect the interaction with Fc receptors and Fc protection receptors, leading to a synergistic effect. The invention also provides fusion proteins with improved serum half-life and methods for optimizing the interaction with Fc receptors and Fc protection receptors.

Problems solved by technology

However, the utility of recombinantly-produced antibody-based fusion proteins may be limited by their rapid in vivo clearance from the circulation.

Method used

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  • Enhancing the circulating half-life of antibody-based fusion proteins
  • Enhancing the circulating half-life of antibody-based fusion proteins
  • Enhancing the circulating half-life of antibody-based fusion proteins

Examples

Experimental program
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Effect test

example 1

Construction of Antibody-IL-2 Genes with Substitutions of the Lys Codon at the Fusion Junction

[0057] The amino acid sequence at the junction of the antibody-IL-2 fusion protein is SerProGlyLys-AlaProThr (SEQ ID NO: 1), in which the SerProGlyLys (SEQ ID No. 2) is the normal carboxy terminus of the heavy chain of the antibody, and AlaProThr is the N-terminal sequence of mature IL-2. In order to determine the effect alterations in the region of the fusion junction on the pharmacokinetics of the fusion protein, substitutions or deletion of the residue were made by mutagenesis, as described below.

[0058] The expression vector for immunocytokines was described in Gillies at al., (1998) J. Immunol. 160:6195-6203. In the human gamma-1 gene encoding the heavy chain, the XmaI restriction site located 280 bp upstream of the translation stop codon was destroyed by introducing a silent mutation (TCC to TCA). Another silent mutation (TCT to TCC) was introduced to the Ser codon three residues ups...

example 2

Construction of Antibody-IL-2 Genes Encoding Extra Amino Acid Residues at the Fusion Junction

[0069] It is common in the art to separate domains in fusion proteins with flexible linkers containing amino acid residues such as glycine and serine. The importance of the spacing between the CH3 and IL-2 was studied in the following mutagenesis experiments. Blunt ended oligonucleotide duplexes encoding different number of amino acid residues were inserted into the SmaI endonuclease restriction site (same recognition site as the XmaI mentioned above) of the huKS-IL-2 expression vector by ligation; and the correct orientation of insertion was confirmed by DNA sequencing. As discussed above, oligonucleotide duplexes with 5′-hydroxyl ends were used to eliminate self ligation.

9.) Lys to Cys Substitution with Linker Ligation

[0070] The following linker (oligonucleotide duplex) was inserted into the SmaI endonuclease restriction site of the huKS-IL-2 expression vector by ligation. The sequence...

example 3

Construction of Antibody-IL-2 Genes with Substitutions of the Pro Codon at the Fusion Junction

[0074] The proline in the sequence ProGlyLys at the carboxyl terminus of CH3 is mutated to Ala, Leu or Gly, and other amino acids. This is accomplished by replacing a 25 base-pair SapI-SmaI fragment of the KS-IL-2 expression vector by an oligonucleotide duplex encoding the desired change. Each of the following oligonucleotide duplexes has a SapI cohesive end (3-base overhang) and a blunt end (for ligating to the SmaI end of the restriction fragment). The substitutions at the Pro codon are denoted in bold. These substitutions had no significant effect on the pharmacokinetics of the fusion protein, indicating that the structural properties of the Pro residue have no significant effect on the pharmacokinetics of the fusion protein

12.) Pro to Ala Substitution(SEQ ID NO:28)5′ CG CAG AAG AGC CTC TCC CTG TCC GC 3′(SEQ ID NO:29)3′     TC TTC TCG GAG AGG GAC AGG CG 5′13.) Pro to Leu Substitution(...

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Abstract

Disclosed are compositions and methods for enhancing the circulating half-life of antibody-based fusion proteins. Disclosed methods and compositions rely on altering the amino acid sequence of the junction region between the antibody moiety and the fused protein moiety in an antibody-based fusion protein. An antibody-based fusion protein with an altered amino acid sequence in the junction region has a greater circulating half-life when administered to a mammal. Disclosed methods and compositions are particularly useful for reducing tumor size and metastasis in a mammal.

Description

RELATED APPLICATIONS [0001] This application claims priority to, and the benefit of U.S. Ser. No. 60 / 181,768, filed Feb. 11, 2000, the disclosure of which is incorporated by reference herein.FIELD OF THE INVENTION [0002] The present invention relates generally to fusion proteins. More specifically, the present invention relates to methods of enhancing the circulating half-life of antibody-based fusion proteins. BACKGROUND OF THE INVENTION [0003] The use of antibodies for treating human diseases is well established and has become more sophisticated with the introduction of genetic engineering. Several techniques have been developed to improve the utility of antibodies. These include: (1) the generation of monoclonal antibodies by cell fusion to create “hybridomas”, or by molecular cloning of antibody heavy (H) and light (L) chains from antibody-producing cells; (2) the conjugation of other molecules to antibodies to deliver them to preferred sites in vivo, e.g., radioisotopes, toxic ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/395C07K16/44A61K39/44A61K47/48C07K5/103C07K14/47C07K14/52C07K14/525C07K14/55C07K14/705C07K16/00C07K16/18C07K16/30C07K19/00
CPCA61K47/48423A61K47/48569A61K2039/505C07K5/1013C07K14/525C07K14/55C07K2317/52C07K16/30C07K19/00C07K2317/24C07K2317/73C07K2319/00C07K16/00A61K47/6813A61K47/6851
Inventor GILLIES, STEPHENBURGER, CHRISTALO, KIN-MING
Owner MERCK PATENT GMBH
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